Mechanisms of RNA binding and remodeling proteins Abstract Manipulation of RNA requires the action of RNA binding proteins and ATP-dependent, molecular motor proteins that are believed to transport, remodel, and unwind secondary structures in RNA sequences. Many of these molecular motors are DEAD-box or closely related proteins. Positive strand RNA viruses such as the Hepatitis C virus (HCV) require the activity of these proteins for viral replication. Determination of the molecular mechanisms of these proteins is of fundamental importance to our understanding of RNA metabolism in general and will advance our understanding of how RNA virus'are replicated. The goal of this project is to determine the mechanism of RNA remodeling by an RNA binding protein and an RNA remodeling enzyme from the Hepatitis C virus. Non-structural protein 3 (NS3) is an RNA remodeling enzyme (or helicase), that is necessary for HCV replication. We propose a new model for RNA remodeling by this enzyme whereby multiple subunits of an oligomeric enzyme work together to melt out secondary structures in RNA. Our model predicts that NS3 participates in slow untwisting of the duplex in slow kinetic step, followed by rapid translocation in an ATP-dependent manner. We will test this model by using a combination of biophysical, biochemical, and biological experiments. We will identify the specific sites of protein-protein interactions by using protein footprinting coupled with mass spectrometry. Truncated forms of NS3 that do not form oligomeric structures will be examined to determine the specific roles that protein-protein interactions play in RNA remodeling. Our work has identified NS5A as an RNA binding protein that interacts with itself and with NS3. The structure of NS5A represents a new fold in nucleic acid recognition, and we are poised to uncover the structure/function relationship for RNA binding by this protein. The importance of protein-protein interactions will be determined by preparing variants of NS5A that are impeded in dimerization, followed by testing of those variants for RNA binding activity and for support of HCV replication in cells. The interplay between the NS3 and NS5A will be examined in detail using new biochemical and biological approaches. In aim 1, we will test our hypothesis for RNA remodeling by NS3. In aim 2, we will determine the role of dimerization of NS5A in RNA binding. In aim 3, the we will test the hypothesis that NS5A serves as a processivity factor for NS3 helicase activity. Determining the mechanisms of HCV RNA binding and remodeling proteins will reveal new molecular methods to disrupt the pathways responsible for HCV replication. Therefore, understanding the molecular mechanisms of proteins that bind and manipulate RNA is of biological and medical significance. PUBLIC HEALTH RELEVANCE: Mechanisms of RNA binding and remodeling proteins Narrative: Proteins that bind and manipulate RNA are of fundamental importance to many biological processes including translation, transcription, and gene regulation. RNA binding protiens also play key roles in viruses such as the Hepatitis C Virus, which infects almost 3 % of the world's population. Over 75% of HCV infections never resolve, resulting in persistent virus infection that can lead to liver fibrosis and, progressively, to severe and fatal diseases, including liver cirrhosis and liver cancer. Determining the mechanisms of HCV RNA binding and remodeling proteins will reveal new molecular methods to disrupt the pathways responsible for HCV replication. Therefore, understanding the molecular mechanisms of proteins that bind and manipulate RNA is of general biological and medical significance.